BIOCHEMICAL PROPERTIES OF SOMATOSTATIN RECEPTORS

生长抑素14(SRIF)是中枢神经系统的一种神经递质。 它已被证明在大脑中有许多功能，包括 调节神经元放电和神经递质释放。该多肽可以 在某些行为的表达上也起着作用，比如认知。 SRIF在大脑中传递的异常与一种 各种精神障碍，如精神分裂症、抑郁症和 阿尔茨海默氏症。尽管SRIF作为一种神经递质很重要， 关于这种多肽是如何诱导其生理效应的，人们知之甚少。 这项提议的目的是为了更好地理解分子 SRIF在脑内的作用机制。这将通过以下方式实现 鉴定SRIF受体的物理性质。通过识别 SRIF受体的性质，我们可能会深入了解 受体与不同的转导系统相互作用，如腺苷 循环酶系统和离子电导通道。为了调查 SRIF受体的生化性质，我们将纯化大鼠的大脑 SRIF受体使用亲和层析技术。为了证明 纯化的蛋白是SRIF受体，我们将尝试 用SRIF类似物[125I]CGP 23996特别标记蛋白质。 也将尝试恢复净化的能力 受体介导SRIF刺激GTP酶活性或抑制SRIF活性 磷脂小泡或细胞中腺苷环化酶活性的缺失 在SRIF受体中。一旦纯化的蛋白质被确定为 SRIF受体，该受体的一些物理性质将是 评估过了。受体的大小和电荷将用凝胶测定。 电泳法。受体的低聚糖含量 将同时使用糖酵解酶和凝集素进行研究 层析法。为了进一步描述SRIF的属性 受体，则会产生针对该受体的抗体。生成 抗体，纯化的SRIF受体将被部分测序 使用气相微测序仪。序列信息将用于 合成受体的多肽片段。多克隆抗体将是 针对这些多肽产生的。如果选择性相互作用的抗体 随着SRIF受体的开发，我们将使用这些抗体来 进一步检测脑组织SRIF受体的生化特性 以确定各亚型之间是否存在生理差异 SRIF受体。有功能证据表明SRIF受体亚型 存在于大脑中，与SRIF及其前激素有不同的亲和力 生长抑素28.证明这些受体亚型在生理上 DISTINCT将支持SRIF和生长抑素28是 不同的神经递质。生长抑素28的结构差异 和SRIF受体将在两个细胞系中进行研究，这两个细胞系 表达一种或另一种受体亚型。大小，电荷，肽图， 这些受体亚型所经历的糖含量和加工事件 将会被检验和比较。

Somatostatin 14 (SRIF) is a neurotransmitter in the central nervous system. It has been shown to have a number of functions in the brain including regulating neuronal firing and neurotransmitter release. The peptide may also play a role in the expression of certain behaviors such as cognition. Abnormalities of SRIF transmission in the brain have been implicated in a variety of mental disorders such as Schizophrenia, depression and Alzheimer's disease. Despite the importance of SRIF as a neurotransmitter, little is known about how this peptide induces it's physiological effects. The objective of this proposal is to better understand the molecular mechanisms of action of SRIF in brain. This will be accomplished by identifying the physical properties of the SRIF receptor. By identifying the properties of the SRIF receptor we may gain insight into how the receptor interacts with different transducing systems such as the adenylate cyclase system and ionic conductance channels. To investigate the biochemical properties of the SRIF receptor, we will purify the rat brain SRIF receptor using affinity chromatographic techniques. To demonstrate that the purified protein is the SRIF receptor, we will attempt to specifically label the protein with the SRIF analogue [125I] CGP 23996. Attempts will also be made to reconstitute the ability of the purified receptor to mediate SRIF stimulation of GTPase activity or SRIF inhibition of adenylate cyclase activity in phospholipid vesicles or cells deficient in the SRIF receptor. Once the purified protein is established to be the SRIF receptor, some of the physical properties of the receptor will be assessed. The size and charge of the receptor will be determined using gel electrophoretic techniques. The oligosaccharide content of the receptor will be investigated using both glycolytic enzymes and lectin chromatography. To further characterize the properties of the SRIF receptor, antibodies will be generated against the receptor. To generate the antibodies, the purified SRIF receptor will be partially sequenced using a gas phase microsequenator. The sequence information will be used to synthesize peptide fragments of the receptor. Polyclonal antibodies will be generated against these peptides. If antibodies that selectively interact with the SRIF receptor are developed, we will use these antibodies to further examine the biochemical properties of brain SRIF receptors as well as to determine whether there are physical differences between subtypes of SRIF receptors. There is functional evidence that SRIF receptor subtypes exist in brain with differing affinities for SRIF and its prohormone somatostatin 28. Demonstration that these receptor subtypes are physically distinct would support the notion that SRIF and somatostatin 28 are distinct neurotransmitters. Differences in the structure of somatostatin 28 and SRIF receptors will be investigated in two cell lines which exclusively express one receptor subtype or the other. The size, charge, peptide maps, sugar content and processing events which these receptor subtypes undergo will be examined and compared.